Mixing and matching light heads and filters

 

The light sources and barrier filters for the Model SFA Stereo Microscope Fluorescence Adapter and the Xite flashlight packages are packaged in sets that are generally used together. It is possible to use the light head from one set with the barrier filter from another set to solve some viewing problem, as in the example described here:

Dr. Martha Marvin of Williams College uses the SFA system to look at transgenic zebrafish (Dania rerio) with several different fluorescent proteins (see reference at bottom of page). She wrote:

“We have fish with blue fluorescent (BFP) hearts that also up-regulate GFP in response to stress. To sort the transgenics we look at blue fluorescence. That didn’t work well with the violet barrier filter, but if we illuminate with violet and view through the royal blue barrier filter it’s quite easy to see.”

The blue fluorescence came from Evrogen TagBFP. Based on the published spectra for this fluorophore one would expect our Violet light + filter set (400 – 415nm excitation, 460nm longpass emission) to be the best choice for exciting and capturing the most fluorescence. However in this case there was some general blue fluorescence in the background that made it hard to reliably distinguish the blue fluorescence of the hearts. This background fluorescence did not pass through the yellow filter that comes with the Royal Blue filter set, while enough of the BFP emission did, resulting in improved contrast.

Reference – Krug, R. G., Poshusta, T. L., Skuster, K. J., Berg, M. R., Gardner, S. L., & Clark, K. J. (2014). A transgenic zebrafish model for monitoring glucocorticoid receptor activity. Genes, Brain and Behavior, 13(5), 478-487.

 

These two bullets summarize the rules for combining light heads and filters.

  • For any excitation light head you can pair it with any of the barrier filters that is normally used with a light head of a longer wavelength.
  • For any barrier filter, you can pair it with any of the light heads that is normally used with a barrier filter of a shorter wavelength.

This table summarizes the standard light source / filter combinations:

Designation Excitation Emission
UV – Ultraviolet 360 – 380nm 415nm longpass
VI – Violet 400 – 415nm 450nm longpass
RB – Royal Blue 440 – 460nm 500nm longpass
RB – Royal Blue 440 – 460nm 500 – 560nm bandpass
CY – Cyan 490 – 515nm 550nm longpass
GR – Green 510 – 540nm 600nm longpass

 

The possible combinations are shown in this table, with permissible pairings highlighted in green, forbidden combinations in red.

Barrier Filters
Light Heads UV  VI  RB RB-GO CY GR
UV
VI
RB
CY
GR